This invention relates to substrates for use in the fluorescent, phosphorescent or luminescent detection of biological or chemical samples. Fluorescence microscopy is often used in the fields of molecular biology, biochemistry and other life sciences for analyzing biological molecules, including nucleic acids (DNA, RNA) and proteins (enzymes, antigens, etc.) that have been tagged or labeled with fluorescent probes. One such use is DNA diagnostics, such as for gene detection, in which a DNA sample is deposited on and bound to a glass substrate. The bound DNA on the substrate can then be imaged by fluorescence. The fluorescence of a sample has in the past been manually assessed by visual inspection through a conventional microscope, but this manual method is time-consuming and costly. As an improvement, various automated fluorescence imaging systems are now available. However, the structures employed to provide an observation platform fail to take full advantage of the characteristics of the fluorescent probes.
An important aspect of fluorescence detection and measurement instruments is sensitivity, which is primarily determined by the signal-to-noise ratio (SNR) of the optical imaging system of the instrument. One obvious approach to increasing SNR, and thereby improving sensitivity, is to reduce background noise. Sources of background noise include specular or diffuse reflection of the fluorescence-stimulating laser light from the sample, autofluorescence of the substrate holding the sample, autofluorescence from the optics in the light path of the optical imaging system, stray light, and dark current of the detector. Both stray light and much of the reflected laser light can be rejected, while passing the fluorescent light, by using dichroic and other spectral filters and beam splitters in the system.
More recent approaches for enhancing detection involve the use of improved substrate surfaces. U.S. Pat. No. 6,008,892 to Kain et al. and WO 02/48691 to Chaton et al. disclose sample substrates which are reflective for specific excitation wavelengths. U.S. Pat. No. 7,227,633 to Kraus et al., further develops such technology by providing substrates with enhancement of the fluorescence signal for two or more different excitation wavelengths. However, each of these disclosures describes optimized substrates for the detection of samples that are at or substantially near the surface of the substrate. Detection using these substrates does not take into account the finite thickness of certain imaging samples, and thus are not optimized for samples in which the plane of maximum fluorescent signal differs from the surface.
It is helpful to understand certain terms of art. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art. The following definitions are intended to aid in understanding the present invention but are not intended to vary or otherwise limit the meaning of such terms unless specifically indicated.
“Fluorophores” are any molecule comprising or consisting of a functional group that absorbs energy within a specific absorption spectrum and re-emits energy at a different (but equally specific) emission spectrum. Preferred fluorophores for use as markers in the present invention include, but are not limited to, fluorescein, cascade blue, hexachloro-fluorescein, tetrachloro-fluorescein, TAMRA, ROX, FAM, Cy3, Cy3.5, Cy5, Cy5.5, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5-styryl-4-bora-3a,4-adiaz-a-S-indacene-propionic acid, 6-carboxy-X-rhodamine, N,N,N′,N′-tetramethyl-6-carboxyrhodamine, Texas Red, Eosin, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-ethoxyphenyl-4-bora-3a,4a-diaza-s-indacene 3-propionic acid and 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-5-indacene-ptopionic acid, the DyLight Fluor family available from Thermo Fisher Scientific of Waltham, Mass. and the Alexa Fluor family from Molecular Probes of Eugene, Oreg.
The terms “intensity maximum” and “intensity minimum” refer to specific intensities of the radiation (or light) pattern produced by the interference of incident light provided by an outside source with the light reflected from a substrate surface. As used in the context of the present invention detection, “intensity maximum” means at or substantially near the peak of the intensity profile of the radiation (or light) pattern. The term “intensity minimum” used in the context of fluorophore detection means at or substantially near the trough of the intensity profile of the radiation (or light) pattern.
“Solid support” and “support” are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many embodiments, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate regions on the support with, for example, wells, raised regions, pedestals, etched holes, or the like. Microarrays usually comprise at least one planar solid phase support, such as a glass microscope slide.